Glycolic acid fermentative production with a modified microorganism

Abstract

The present invention is related to a method for the fermentative production of glycolic acid, its derivatives or precursors, comprising the culture of an Escherichia coli strain in an appropriate culture medium comprising a carbon source, and the recovery of glycolic acid in the medium, wherein said E. coli strain is modified to improve the conversion of orotate into orotidine 5′-P. The invention is also related to the modified E. coli strain, showing an improved conversion of orotate into orotidine 5′-P, and optionally that was furthermore modified for an improved glycolic acid production.

1 .- 19 . (canceled) 
     
     
         20 . A method for fermentative production of glycolic acid, and/or a derivative or precursor thereof, comprising:
 culturing an  Escherichia coli  strain in an appropriate culture medium comprising a carbon source, and   recovering produced glycolic acid in the medium,   wherein said  Escherichia coli  strain is modified to improve conversion of orotate into orotidine 5′-Phosphate.   
     
     
         21 . The method of  claim 20 , wherein the modified strain presents an increased orotate phosphoribosyl transferase specific activity. 
     
     
         22 . The method of  claim 21 , wherein in said modified strain, expression of gene pyrE encoding orotate phosphoribosyl transferase enzyme is increased. 
     
     
         23 . The method according to  claim 20 , wherein the strain is an  E. coli  K12 strain having a frameshift mutation in the rph-pyrE operon, modified to restore expression of the gene pyrE. 
     
     
         24 . The method according to  claim 20 , wherein the modified strain presents an increased availability of 5-Phosphoribosyl 1-pyrophosphate (PRPP). 
     
     
         25 . The method of  claim 24 , wherein in said modified strain, expression of gene prsA encoding phosphoribosylpyrophosphate synthase is increased. 
     
     
         26 . The method according to  claim 20 , wherein the modified strain is further modified to enhance the production of glycolic acid. 
     
     
         27 . The method according to  claim 26 , wherein the modified strain comprises at least one of the following modifications:
 decrease in conversion of glyoxylate to products other than glycolate,   inability to substantially metabolize glycolate,   increase of glyoxylate pathway flux,   increase in conversion of glyoxylate to glycolate,   increase in availability of NADPH.   
     
     
         28 . The method according to  claim 27 , wherein the modified strain comprises at least one of the following modifications:
 attenuation of the genes aceB, gleB, gel, eda, attenuation of the genes glcDEFG, aldA,   attenuation of the genes icd, aceK, pta, ackA, poxB, iclR or fadR, and/or overexpression of the gene aceA   overexpression of the genes yedW or yiaE   attenuation of the genes pgi, udhA, edd.   
     
     
         29 . The method according to  claim 20 , wherein the carbon source is at least one selected from group consisting of glucose, sucrose, monosaccharides, oligosaccharides, starch, and glycerol. 
     
     
         30 . The method of  claim 20 , comprising:
 a) Fermentation of the modified strain producing glycolic acid   b) Concentration of glycolic acid in bacteria or in the medium and   c) Isolation of glycolic acid from fermentation broth.   
     
     
         31 . The method of  claim 30  wherein glycolic acid is isolated through polymerization to at least glycolate dimers and recovered by depolymerisation from glycolate dimers, oligomers and/or polymers. 
     
     
         32 . An  Escherichia coli  strain, wherein said strain is modified to improve the conversion of orotate into orotidine 5′-Phosphate. 
     
     
         33 . The modified strain of  claim 32 , wherein the strain presents an increased orotate phosphoribosyl transferase specific activity. 
     
     
         34 . The modified strain of  claim 33 , wherein expression of the gene pyre encoding the orotate phosphoribosyl transferase enzyme is increased. 
     
     
         35 . The modified strain of  claim 32 , wherein the strain is an  E. coli  K 12 strain having a frameshift mutation in the rph-pyrE operon, and has been modified to restore expression of the gene pyrE. 
     
     
         36 . The modified strain of  claim 32 , wherein the strain presents an increased availability of 5-Phosphoribosyl 1-pyrophosphate (PRPP) as compared with an unmodified strain. 
     
     
         37 . The modified strain of  claim 36 , wherein expression of the gene prsA encoding phosphoribosylpyrophosphate synthase is increased as compared with an unmodified strain. 
     
     
         38 . The modified strain of  claim 32 , wherein the strain is further modified to enhance production of glycolic acid. 
     
     
         39 . The modified strain of  claim 38 , wherein the modified strain comprises at least one of the following modifications:
 decrease in conversion of glyoxylate to products other than glycolate,   inability to substantially metabolize glycolate,   increase of glyoxylate pathway flux,   increase in conversion of glyoxylate to glycolate,   increase in availability of NADPH.   
     
     
         40 . The modified strain of  claim 39  wherein the modified strain comprises at least one of the following modifications:
 attenuation of the genes aceB, glcB, gcl, eda, 
 attenuation of the genes glcDEFG, aldA, 
 attenuation of the genes icd, aceK, pta, ackA, poxB, iclR or fadR, and/or overexpression of the gene aceA, 
 overexpression of the genes ycdW or yiaE, 
 attenuation of the genes pgi, udhA, edd.